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Proteomics

Scanning and Analysis

Protocols

Prepared by: Bob Morrison

STLCC-CPLS, Instrumentation Specialist

Dec 08, June 2013 (Densitometer offline)

Discover, Quality One Software

NCI Flicker Website

BioRad/UMAX GS-800

Densitometer

Gelscape Analysis Website

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Proteomics : BioRad GS-800 Densiometer Basic Features

Link to BioRad/UMAX GS-800 Users Manual (pdf)

The GS-800 calibrated densitometer delivers automatic calibration for superior accuracy, resolution, and

image quality. With a 30 x 40 cm (11 x 16") imaging platen, 12-bit data collection, and resolution down to

36.3 µm

Automated self-calibration for accurate quantitation

Variable scanning resolution for optimum image acquisition

12-bit (4,096 gray levels) data collection for greater linearity and sensitivity

Dynamic range from 0 to 3.0 OD

Scanning white light (400–750) nm illumination for high sensitivity

Transmittance and reflectance collection for imaging a wide variety of samples

Powerful, user-friendly Quantity One software for image acquisition and data analysis

Sealed platen for imaging of wet samples

Modified transparent lid for safely imaging samples of variable thickness

Provides quantitatively accurate, high-quality imaging to detect and resolve even the faintest

bands. With user-selectable scan resolution down to 36.3 µm, you can preview and scan your

samples in seconds. Variable resolution also allows you to control both the scanning time and

image file size.

High sensitivity. The color CCD camera can scan in red, green, or blue, and scans virtually any

sample (1-D and 2-D gels, colorimetric dot and slot blots, film-based chemiluminescent blots,

autoradiograms, slides, and photographs) at the ideal wavelength for greatest sensitivity and

accuracy.

Has both transmittance and true reflectance capabilities that allow accurate scans of both

transparent and opaque samples. The advantage of true reflectance scanning is the accurate

detection and analysis of molecules on the surface of membranes, rather than molecules

distributed throughout the membrane matrix. True reflectance provides the most accurate

detection and analysis of dot blots, slot blots, and other electrophoretic blots without

quantitation errors.

Densitometer Offline June 2013

The Densitometer software does not run on the

standard Windows operating system used on newer

PCs, therefore it has been disconnected from the

Imager host PC.

As a backup, an older PC is stored below in the

cabinet and can be configured for use of the

Densitometer. Please contact Bob Morrison in R123

or by phone (314-971-3795) for assistance in setting

this up.

Proteomics: Densitometer-Acquire Image- Quality One

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1. Turn on Densitometer/Scanner and

wait for both lights to show steady

green- this is the ready state.

2. Turn on Host computer, logon as

Operator/SMET

3. Select Quality One to scan (acquire)

and image

Link to BioRad Quantity One Software Users Manual (pdf)

Proteomics: Quality One-Select Scanner

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1. Choose Select Scanner on the

Volume Quick Guide menu

2. Select GS-800 as scanner type

Proteomics: Quality One- Scan-Step I-Select Image Type

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1. Step I, Select image and then stain types

2. Step II, Preview Scan and wait for image

outline to appear.

3. Hit STOP if image preview fails to appear

Proteomics: Scan- Step II-Select Scan Area, Step III –Select Resolution

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1. Drag box to outline scan image

2. Step III, change scan resolution in

pop-up box if required

Proteomics: Scan- Acquire Image, wait for scan to complete

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Scanning; Wait for Scan in to proceed

Proteomics: Quality One Densitometer- Scan-Save Image

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To save scanned image select File, then

Save-as, then change filename, and

Save

Proteomics: Quality One - Image-Analysis-Lanes-Volumes

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Follow this link to clarify lanes and identify purity on your scanned image.

Link to Bio-Rad “Quality One” Lane Analysis Tutorial (pdf)

Follow this link to identify volumes (quantitation) for unknowns and

standards on your scanned image and to generate reports of both:

Link to Bio-Rad “Quality One” Volume Analysis Tutorial (pdf)

Lanes

Volumes

Proteomics: Image-Gel Issues (Examples)

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Follow this link to see examples and causes of common gel issues.

Link to Bio-Rad “Gel Clinic” pdf)

Proteomics: SDS_PAGE

Examples

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Proteomics: Gelscape- Enter Username, Password

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http://www.gelscape.ualberta.ca:8080/htm/index.html

Username: Ktoal

Password: 17gelscape

Link to Gelscape

U. Of Alberta Canada

Proteomics: Gelscape-Window Size-Annotate & View Panel

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Adjust Window size to

Enlarge annotation panel

Proteomics: Gelscape-Browse to find your Image

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Browse to locate

your image (gif, jpg)

Proteomics: Gelscape- Morph & Compare

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Browse to locate

your image (gif, jpg)

Proteomics: Gelscape –GelBank- Other image sources

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Search in Gelscape or

other databases

Proteomics: Gelspace- Access other 2D images

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Link to NCI

Flicker Website

Proteomics : BioRad Densiometer; 2D Gel Flicker Website

Link to

http://www.ccrnp.ncifcrf.gov/flicker/indexFrmToc.html

Proteomics: 1D PDQuest-Help-Register-Enter Password 1/26/09 RGM

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Proteomics: Gel Electrophoresis Background

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Gel Electrophoresis: Gel Electrophoresis is a separation technique that is used to separate macromolecules such as nucleic acids

or proteins on the basis of size, electric charge, and other physical properties.. It can be performed within one dimension, two

dimensions, or in a capillary. The sample is mixed into a buffer, and run on gels. Electrophoresis separations are usually carried out

on gels made of agarose or polyacrylamide. These gels are chemically inert, so they will interfere little with the molecules. When

electrophoresis is complete, the gel is stained to make the proteins visible. Common dyes used are Coommasie blue or silver

staining. This method of protein separation and identification is useful because very little protein is needed to determine a difference

in a protein. Distinct spots can be found with as little as 0.1 mg of protein when stained with Coommasie blue and even less (0.02

mg), is need when silver staining. Other methods of staining are Fluorescent dyes, and zinc or copper staining. Some drawbacks to

this technique are that it is time-consuming and the purity of your protein sample will affect your results.

One-Dimensional: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) - this method separates based on the

mass of the molecule. Sodium dodecyl sulfate (SDS) is a detergent that breaks up the interactions between proteins. The proteins

are dissolved in SDS and then electrophorised. The smallest molecules move through the fastest, while larger molecules take longer

and result in bands closer to the top of the gel.

Isoelectric Focusing(IEF) - this method separates based on the isoelectric point(pI) of the protein. The isoelectric point is when the

protein carries no net charge. The protein sample is applied to an immobilized pH gradient(IPG) strip. IPG strips come available over

different lengths and pH ranges. As the sample is electrophorised, the proteins will migrate toward either the anode or cathode,

depending on charge. The proteins will stop when they reach their respective pI, which results in a band. The band corresponds to a

protein.

Two-Dimensional : 2D Electrophoresis is a technique that blends the two methods listed in One-Dimensional analyses. It is a very

efficient separation technique for proteins. The protein sample is first run on an IPG strip, which after reaching completion is placed in

either a horizontal or vertical SDS gel. This technique results in gels that contain spots. Each spot on the gel corresponds to a

different protein. Then the gels are stained similarly as to 1D analysis. 2D Electrophoresis is widely used, and certain methods of it

can be coupled with mass spectrometry in order to identify proteins.fgfrgcfsdfdfs

Capillary: Capillary Electrophoresis is very similar to 1D or 2D Electrophoresis, except it is performed within the small space of a

capillary tube. This is advantageous in many ways. The heating that takes place due to high voltage loads on slab gels can have a

negative effect on the separation of the proteins and the use of capillaries lessens said heat build-up. Also, because the gel will not

need to be handled, it can allow one to use liquid polymers for separation, and can be replaced between runs. Automation is also

much more possible with this technique, lessening the time, and making reproducible results.

Retrieved from "http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/What_is_Electrophoresis%3F"

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Proteomics : Themo Chiller for Gel Supercooling Coil

(example only not purchased)

NESLAB RTE-7 Digital Plus Refrigerated Bath

271103200000-00i

$2,957.00

The RTE-7 is the perfect partner for most CCC applications requiring temperature regulation.

This refrigerated/heated circulating bath offers the highest cooling capacity per dollar, a small

footprint to conserve valuable bench space, and a 2 year warranty for peace of mind.

Temperature Range :-25°C to +150°C

Cooling Capacity :500 Watts at 20°C fluid temperature

Pump Flow :15 liters per minute maximum

Pump Pressure:16 feet maximum

Pump Type:Force and Suction

Heater Wattage :800 Watts (2000 Watts 230 volt models)

Temperature Stability :+/-0.01°C

Reservoir Volume:7 liters Reservoir

Dimensions (WxLxD) :6.6" x 7.2" x 6" (16.8cm x 18.3cm x 15.2cm)

Unit Dimensions (HxWxD) :23 5/8" x 9 1/4" x 17 1/2" (60cm x 23.5cm x 44.5cm)

Shipping Weight :63 lbs. (28.6 kgs)

The Thermo Scientific NESLAB RTE-7 7 liter refrigerated circulating bath offers great value

for either external circulation or in-bath applications:

· Large work openings for big or multiple samples

· Advanced features to further optimize your system available with the Digital Plus

· Wide temperature range and high cooling capacities (500W) to meet your application needs

(down to -25°C)

The super cooling coil and a water re-circulator maintain constant buffer

temperature and prevent buffer depletion during native enzyme, high-

intensity, or overnight transfers